CN106771170A - A kind of colloidal gold fast detecting reagent kit of soybean medicinal extract Daidzein - Google Patents
A kind of colloidal gold fast detecting reagent kit of soybean medicinal extract Daidzein Download PDFInfo
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- CN106771170A CN106771170A CN201710044716.5A CN201710044716A CN106771170A CN 106771170 A CN106771170 A CN 106771170A CN 201710044716 A CN201710044716 A CN 201710044716A CN 106771170 A CN106771170 A CN 106771170A
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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Abstract
The invention particularly discloses a kind of colloidal gold fast detecting reagent kit of soybean medicinal extract isoflavones, including cassette holder, lid and test strips, the lid is provided with well and observation panel, the test strips, the lid and cassette holder clamping are fixed with the cassette holder, the test strips include sample paper, all-glass paper, nitrocellulose membrane and blotting paper, the sample paper, all-glass paper, nitrocellulose membrane and blotting paper are from left to right arranged, and are connected successively.Kit detection time in the present invention is fast, convenient, stabilization and accurate, is adapted to scope wide.Detection method of the invention, it is not necessary to the pretreatment of complexity is carried out to sample, operating procedure is brief, Large expensive instrument need not be used, do not limited by laboratory apparatus condition, being conducive to the scene of detection is carried out, the grass-roots unit for being conducive to Setup Experiments to fall behind carries out experiment.
Description
Technical field
The invention belongs to drug measurement techniques field, in particular it relates to a kind of glue of soybean medicinal extract Daidzein
Body gold quick detection kit.
Background technology
Soybean is a kind of important oil crops, is the crop soybean nutritional that a kind of its seed contains abundant phytoprotein
Comprehensively, the content of rich content, wherein protein is higher than pork 2 times, is 2.5 times of egg content.The content of protein is not only
Height, and quality is good.Soybean is the most frequently used to be made various bean product, squeezes soya-bean oil, makes soy sauce and extract protein, can also be carried
Refining isoflavones.
Daidzein also known as daidzein, are one kind of isoflavones, are obtained by being extracted in legume, Daidzein
Can be used for hypertension, symptomatic hypertension, coronary heart disease, cerebral thrombus, myocardial infarction, arrhythmia cordis, climacteric metancholia of women etc.
Treatment.Daidzein molecular formula is C15H10O4, molecular weight：254.24.
At present, the method for detection Daidzein has high performance liquid chromatography (HPLC), high performance liquid chromatography-tandem mass method
(LC-MS-MS) and the instrumental method such as capillary electrophoresis (HPCE), but these methods exist cumbersome, it is right to be needed before detection
Sample carries out the pretreatment of complexity；Time-consuming；The instrument of Large expensive is needed to use, the shortcomings of expense is somewhat expensive, it is impossible to realize
The quick detection of a large amount of samples.Therefore a kind of sensitivity of urgent need exploitation is high, and detection speed is fast, the detection method of high specificity.
The content of the invention
For the deficiency that above-mentioned prior art is present, tried the invention provides a kind of detection of agricultural product medicament residue collaurum
Agent box, to solve above-mentioned technical problem.
The present invention is achieved through the following technical solutions：
A kind of colloidal gold fast detecting reagent kit of soybean medicinal extract Daidzein, including cassette holder, lid and test paper
Bar, the lid is provided with well and observation panel, and the test strips, the lid and cassette holder card are fixed with the cassette holder
Connect, the test strips include sample paper, all-glass paper, nitrocellulose membrane and blotting paper, the sample paper, all-glass paper,
Nitrocellulose membrane and blotting paper are from left to right arranged, and are connected successively；Detection extract of soybean is coated with the all-glass paper
Gold labeling antibody；The envelope antigen of detection extract of soybean is coated with the nitrocellulose filter, the detection is marked with gold
Antibody can with detection envelope antigen association reaction and develop the color, and only one of which binding site in detection gold labeling antibody.
Preferably, second IgG of animal protein is also coated with the plain film of the dimension, it can be detected with phthalic acid
With gold labeling antibody association reaction and develop the color.
Preferably, the envelope antigen of the detection extract of soybean is prepared from for its haptens.
Compared with prior art, the invention has the advantages that：
1st, the kit detection time in the present invention is fast, convenient, stablize and accurately, be adapted to scope wide.
2nd, detection method of the invention, it is not necessary to the pretreatment of complexity is carried out to sample, operating procedure is brief, it is not necessary to make
Large expensive instrument is used, is not limited by laboratory apparatus condition, being conducive to the scene of detection is carried out, be conducive to what Setup Experiments fell behind
Grass-roots unit carries out experiment.
4th, the testing result and accuracy of kit of the invention HPLC methods testing result higher closely, kit
Testing result and legitimate reading accuracy controlling controlled 12% in the Parallel testing result average deviation of 95% or so, 5 time
Within, illustrate that the kit testing result degree of accuracy of the invention is higher, the colloid of live a large amount of sample screenings can be met substantially
Gold reagent box.
Brief description of the drawings
Fig. 1 is colloidal gold fast detecting reagent kit lid structural representation of the invention.
Fig. 2 colloidal gold fast detecting reagent kit cassette holder structural representations of the invention.
Fig. 3 is test strips structure schematic diagram of the invention.
Wherein：Well 1-1, observation window 1-2, product paper 3-1, cassette holder 2, sample paper 2-1, all-glass paper 2-2, nitric acid are fine
Dimension plain film 2-3, blotting paper 2-4, glass fibrous paper 3-2, nitrocellulose filter 3-3, detection line 3-31, nature controlling line 3-32 blotting papers 3-
4。
Specific embodiment
The invention will be further described with the following Examples.
A kind of colloidal gold fast detecting reagent kit of soybean medicinal extract Daidzein, including cassette holder, lid and test paper
Bar, the lid is provided with well and observation panel, and the test strips, the lid and cassette holder card are fixed with the cassette holder
Connect, the test strips include sample paper, all-glass paper, nitrocellulose membrane and blotting paper, the sample paper, all-glass paper,
Nitrocellulose membrane and blotting paper are from left to right arranged, and are connected successively；Detection extract of soybean is coated with the all-glass paper
Gold labeling antibody；The envelope antigen of detection extract of soybean is coated with the nitrocellulose filter, the detection is marked with gold
Antibody can with detection envelope antigen association reaction and develop the color, and only one of which binding site in detection gold labeling antibody.
Preferably, state and be also coated with nitrocellulose filter second IgG of animal protein, it can be with phthalic acid
Detection gold labeling antibody association reaction simultaneously develops the color.
The envelope antigen is Daidzein comlete antigen.
Preferably, the envelope antigen of the detection extract of soybean is prepared from for its haptens.
Daidzein belongs to small-molecule substance, itself is not available for the active function groups (such as amino, carboxyl) of coupling,
Therefore the antigen that coupling reaction produces specific antibody cannot occur with carrier protein, it is impossible to which direct induced animal body produces special
Heterogenetic antibody, it is therefore desirable to carry out modification derivatization to Daidzein molecule, connecting active function groups can be with carrier protein
Coupling is then likely to form antigen, and this antigen after manually modified is referred to as artificial antigen, and the derivatization product then claims
It is haptens.
The preparation method of Daidzein comlete antigen is below will be described in detail, preparation process is comprised the following steps：
(1) Daidzein haptens；
(2) Daidzein comlete antigen is prepared with Daidzein haptens.
Preferably, the preparation method of above-mentioned steps (1) Daidzein haptens is comprised the following steps：
S1. Daidzein is dissolved in acetone, sequentially adds potassium hydroxide solution, KI and bromoacetate, room temperature
Lower stirring reaction；
S2. to adding hydrochloric acid solution to be neutralized in the reaction solution of step S1, distilled water is added, is merged after then extracting
Organic layer, dry filter, rotary evaporation removes ethyl acetate, obtains crude product ester；
S3. gained crude product ester acetone is redissolved, and solvent is made with chloroform and methyl alcohol, and thin-layer chromatography is carried out at room temperature, is obtained
Target product ester；
S4. target product ester is dissolved in methyl alcohol, adds potassium hydroxide solution to be reacted；Neutralized with hydrochloric acid solution again,
Distilled water is added, organic layer is merged after then extracting, dry filter, rotary evaporation removes ethyl acetate, obtains target product.
The preparation method step of the Daidzein haptens is simple, and the haptens of synthesis can be used for preparing Daidzein complete
Holoantigen, for the preparation of daidzin antibody provides new approach, safely and effectively, the purity of antibody is also higher for the antibody of preparation.It is full
The need for the foot country is to the research, with wide actual application prospect.
Preferably, the preparation method of above-mentioned steps (2) Daidzein comlete antigen is comprised the following steps：
S1. Daidzein haptens, EDC and NHS are dissolved in the DMF of 200 μ L, reaction is stirred at room temperature overnight, obtain final product work
The Daidzein derivative changed；
S2. carrier protein is dissolved in 0.7% sodium chloride solution, then is added dropwise over the good Huang of above-mentioned activation while stirring
Beans aglycone derivative, reacts 1~5h at room temperature, after reaction terminates, is dialysed with 0.7% sodium chloride solution, that is, obtain daidzin
First comlete antigen, freezen protective is in -20 DEG C after packing.
Wherein it is preferred to, the mass ratio of Daidzein haptens, EDC and NHS in step S1 is 1：0.6~1.2：0.4
~1.
Preferably, the Daidzein haptens in step S1：DMF=7mg/mL.
Preferably, the carrier mass in step S2：Sodium chloride solution=11~20mg, 1.8~2.5mL.
Preferably, carrier mass described in step S2 is protein, protein fragments, synthesis polypeptide, semi-synthetic polypeptide, many
Sugar, such as seralbumin, globulin, lipoprotein, polyamino acid or glucan；Or the carrier mass is active base
Other synthesis of group or natural polymer, such as diphtheria toxin, tetanus toxin, yeast, nylon, dextran or cellulose.
Experimental example
Kit of the invention is contrasted with HPLC methods, detects the content of Daidzein in several samples, and RNA isolation kit is real
Group is tested, HPLC methods are control group, and each treatment is repeated 3 times, and experimental result is averaged, as a result as shown in table 1.
Table 1
| Sample | Experimental group | Control group | 5 result errors |
| Soybean | 5.62ppb | 6.14ppb | 9.6% |
| Black soybean | 12.58 ppb | 11.75 ppb | 8.4% |
| Feed soybean | 19.31 ppb | 20.67 ppb | 11.5% |
Two kinds of detection methods in consolidated statement 1, compare it is known that kit of the invention testing result and accuracy compared with
HPLC methods testing result high closely, the testing result of kit with legitimate reading accuracy controlling 95% or so, 5 times
Parallel testing result average deviation control within 12%, illustrate that the kit testing result degree of accuracy of the invention is higher, base
Originally the colloidal gold kit of live a large amount of sample screenings can be met.
Finally it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention, rather than the present invention is protected
The limitation of scope is protected, although being explained to the present invention with reference to preferred embodiment, one of ordinary skill in the art should
Work as understanding, technical scheme can be modified or equivalent, without deviating from the reality of technical solution of the present invention
Matter and scope.
Claims (4)
1. a kind of colloidal gold fast detecting reagent kit of soybean medicinal extract isoflavones, including cassette holder, lid and test paper
Bar, the lid is provided with well and observation panel, and the test strips, the lid and cassette holder card are fixed with the cassette holder
Connect, it is characterised in that the test strips include sample paper, all-glass paper, nitrocellulose membrane and blotting paper, the sample paper,
All-glass paper, nitrocellulose membrane and blotting paper are from left to right arranged, and are connected successively；Detection is coated with the all-glass paper
The gold labeling antibody of extract of soybean；The envelope antigen of detection extract of soybean is coated with the nitrocellulose filter, it is described
Detection gold labeling antibody can with detection envelope antigen association reaction and develop the color, and only one of which knot in detection gold labeling antibody
Close site.
2. the colloidal gold fast detecting reagent kit of soybean medicinal extract isoflavones according to claim 1, it is special
Levy and be, second IgG of animal protein is also coated with the nitrocellulose filter, it can detect with phthalic acid and use
Gold labeling antibody association reaction simultaneously develops the color.
3. the colloidal gold fast detecting reagent kit of soybean medicinal extract isoflavones according to claim 2, it is special
Levy and be, the envelope antigen Daidzein haptens of the detection extract of soybean is prepared from.
4. the colloidal gold fast detecting reagent kit of soybean medicinal extract isoflavones according to claim 3, it is special
Levy and be, the Daidzein haptens preparation method is comprised the following steps：
S1. Daidzein is dissolved in acetone, sequentially adds potassium hydroxide solution, KI and bromoacetate, stirred at room temperature
Mix reaction；
S2. to adding hydrochloric acid solution to be neutralized in the reaction solution of step S1, distilled water is added, merges organic after then extracting
Layer, dry filter, rotary evaporation removes ethyl acetate, obtains crude product ester；
S3. gained crude product ester acetone is redissolved, and solvent is made with chloroform and methyl alcohol, and thin-layer chromatography is carried out at room temperature, obtains target
Product ester；
S4. target product ester is dissolved in methyl alcohol, adds potassium hydroxide solution to be reacted；Neutralized with hydrochloric acid solution again, then added
Enter distilled water, organic layer is merged after then extracting, dry filter, rotary evaporation removes ethyl acetate, obtains target product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201710044716.5A CN106771170A (en) | 2017-01-21 | 2017-01-21 | A kind of colloidal gold fast detecting reagent kit of soybean medicinal extract Daidzein |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710044716.5A CN106771170A (en) | 2017-01-21 | 2017-01-21 | A kind of colloidal gold fast detecting reagent kit of soybean medicinal extract Daidzein |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101413956A (en) * | 2008-11-08 | 2009-04-22 | 江苏省苏微微生物研究有限公司 | Colloidal gold detection card for rapidly detecting melamine and preparing method thereof |
| WO2011107238A1 (en) * | 2010-03-01 | 2011-09-09 | System Biologie Ag | Test strip for the detection of equol |
| CN103091495A (en) * | 2013-01-16 | 2013-05-08 | 河南知微生物工程有限公司 | Test paper card for quickly detecting residues of fluoroquinolones and preparation method of test paper card |
| CN104914248A (en) * | 2015-07-08 | 2015-09-16 | 河南省农业科学院 | Colloidal gold immunochromatographic test paper capable of quickly detecting SBA (Soybean Agglutinin) and preparation method |
| CN105669628A (en) * | 2016-01-12 | 2016-06-15 | 华南农业大学 | Daidzein semiantigen and complete antigen and preparation method and application |
-
2017
- 2017-01-21 CN CN201710044716.5A patent/CN106771170A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101413956A (en) * | 2008-11-08 | 2009-04-22 | 江苏省苏微微生物研究有限公司 | Colloidal gold detection card for rapidly detecting melamine and preparing method thereof |
| WO2011107238A1 (en) * | 2010-03-01 | 2011-09-09 | System Biologie Ag | Test strip for the detection of equol |
| CN103091495A (en) * | 2013-01-16 | 2013-05-08 | 河南知微生物工程有限公司 | Test paper card for quickly detecting residues of fluoroquinolones and preparation method of test paper card |
| CN104914248A (en) * | 2015-07-08 | 2015-09-16 | 河南省农业科学院 | Colloidal gold immunochromatographic test paper capable of quickly detecting SBA (Soybean Agglutinin) and preparation method |
| CN105669628A (en) * | 2016-01-12 | 2016-06-15 | 华南农业大学 | Daidzein semiantigen and complete antigen and preparation method and application |
Non-Patent Citations (2)
| Title |
|---|
| YUSAKUL,G等: "Preparation and application of a monoclonal antibody against the isoflavone glycoside daidzin using a mannich reaction-derived hapten conjugate", 《PHYTOCHEMICAL ANALYSIS》 * |
| 王贵升: "大豆黄酮的酶联免疫吸附分析方法的建立及其在大鼠血液、鸡蛋与饲料中含量的检测", 《中国优秀博硕士学位论文全文数据库(硕士)农业科技辑》 * |
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Application publication date: 20170531 |